The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

The DNA methyltransferase Dnmt3a suppresses tumorigenesis in models of leukemia and lung cancer. Conversely, deregulation of Dnmt3b is thought to generally promote tumorigenesis. However, the role of Dnmt3a and Dnmt3b in many types of cancer remains undefined. Here, we show that Dnmt3a and Dnmt3b are dispensable for homeostasis of the murine epidermis. However, loss of Dnmt3a-but not Dnmt3b-increases the number of carcinogen-induced squamous tumors, without affecting tumor progression. Only upon combined deletion of Dnmt3a and Dnmt3b, squamous carcinomas become more aggressive and metastatic. Mechanistically, Dnmt3a promotes the expression of epidermal differentiation genes by interacting with their enhancers and inhibits the expression of lipid metabolism genes, including PPAR-γ, by directly methylating their promoters. Importantly, inhibition of PPAR-γ partially prevents the increase in tumorigenesis upon deletion of Dnmt3a. Altogether, we demonstrate that Dnmt3a and Dnmt3b protect the epidermis from tumorigenesis and that squamous carcinomas are sensitive to inhibition of PPAR-γ.

Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons' intrinsic axonal growth competence are not well understood. Here, we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.

Sexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.

5-hydroxymethyl-cytosine (5-hmC) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (ESC) derived from mammalian blastocysts. Recent observations imply that both 5-hmC and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmC, may play an important role in self renewal and differentiation of ESCs. Here we assessed the distribution of 5-hmC in zebrafish and chick embryos and found that, unlike in mammals, 5-hmC is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. However, 5-hmC is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmC enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmC in its regulation.

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